Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
2000-07-14
2002-11-26
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S183000, C424S094400
Reexamination Certificate
active
06485950
ABSTRACT:
FIELD OF INVENTION
The present invention relates to a novel purified isozyme of an autoclavable superoxide dismutase (EC 1.15.1.1; hereinafter, referred to “SOD”), a process for the identification and extraction of the said superoxide dismutase from
Potentilla astrisanguinea Lodd.
variety
argyrophylla
(Wall. ex. Lehm) Griers (hereinafter, referred to Potentilla) belonging to family Rosaceae. The invention also relates to a process for the extraction of the said SOD and its use in preparing many cosmetic, pharmaceutical and food compositions.
BACKGROUND AND PRIOR ART REFERENCES TO THE INVENTION
SOD is present in plant and animal cells to protect the cellular components against the deleterious effects caused by superoxide radical (hereinafter, referred to O
2
−.
). SOD dismutates superoxide radical into hydrogen peroxide and oxygen as per the following chemical reaction:
2 O
2
−.
+2 H
+
=H
2
O
2
+O
2
If O
2
−.
is not removed, it reacts with H
2
O
2
to produce a highly reactive hyroxyl free radical, which causes lipid peroxidation, protein denaturation and DNA mutation. A living system is said to be under oxidative stress, when such active oxygen mediated reactions are not being taken care of by enzyme systems.
SOD is a critical enzyme to manage oxidative stress both in plants and animal systems. Depending upon the co-factor requirements, the SOD can be Mn-SOD (SOD requiring manganese as a co-factor; localised in mitochondria; insensitive to potassium cyanide and hydrogen peroxide), Cu/Zn-SOD (SOD requiring copper and zinc as co-factors; localised in cytoplasm and chloroplast; sensitive to potassium cyanide and hydrogen peroxide) and Fe-SOD (SOD requiring iron as a co-factor; detected in microbes, blue-green algae and in a few species of higher plants).
SOD is also an important enzyme identified for imparting chilling tolerance to the plants and in stresses for example, water stress, low temperature stress, light stress (particularly, high light intensity), salt stress, radiation stress and all other stresses wherein O
2
−.
is generated in excess quantity to damage the system (Foyer, C. H., Descourvieres, P. and Kunert, K. J. 1994. Plant Cell Environ. 17: 507-523; Allen, R. D., 1995. Plant Physiol. 107: 1049-1054).
In pharmaceutical applications, the enzyme has implications in all those diseases wherein O
2
−.
is produced in a quantity so as to cause damage to the system. Hence, SOD in animal system has following implications:
(1) anti-inflammatory agent in wounds, burns etc. (Nimrod, A. Ezov, N., Parizada, L., Weiss, L., Tochner, Z., Slavin, S., Panet, A. and Gorecki, M. In Frontiers of Reactive Oxygen Species in Biology and Medicine (Eds. Asada, K. and Yoshikawa, T.) Excerpta Medica, Amsterdam, 1994, pp. 383-387);
(2) suppressors of asthamatic response (Ihaku, D., Tanimukai, T., Kitada, O., Taniguchi, N., and Sugita. M. In Frontiers of Reactive Oxygen Species in Biology and Medicine (Eds. Asada, K. and Yoshikawa, T.) Excerpta Medica, Amsterdam, 1994, pp. 407-408);
(3) suppressors of side-effects of anti-cancerous agents and in enhancing the life of tumor-bearing animals (Fugimoto, J. In Frontiers of Reactive Oxygen Species in Biology and Medicine (Eds. Asada, K. and Yoshikawa, T.) Excerpta Medica, Amsterdam, 1994, pp. 411-412);
(4) in relieving rheumatoid arthritis, SOD as a drug is administrated intra-articularly (Goebel, K. M. and Storck, U. 1983. Am. J. Med. 74: 124-128).
(5) in reducing the harmful effects of treatment with ionizing radiations (Edsmyr, F., Huber, W. and Menander, K. B. 1976. Curr. Ther. Res. Clin. Exp. 19: 198-211);
(6) in conferring cardiac protection during heart surgery, heart transplantation, kidney transplantation, and during transplantation of other organs such as skin, lung, liver, and pancreas (mentioned in Marklund; Stefan; Edlund; Thomas, 1998. U.S. Pat. No. 5,788,961);
(7) in prolonging the survival of the perfused isolated rabbit cornea (Neuwirth, L. O., and Dikstein, S. 1985. Curr. Eye. Res. 4: 153-154);
(8) in protecting the isolated lens against photo-peroxidation (Varma, S. D. 1982. Ophthalmic Res. 14: 167-175);
(9) injection of SOD is helpful in reducing the frequency of intraventricular brain hemorrhage following hypotension (Ment, L. R., Stewart, W. B., and Duncan, C. C. 1984. J. Neurosurg. 62: 563-569);
(10) in ameliorating hepatitis in rats induced by injection of
Corynebacterium parvum
(Arthur, M. J., Bentley, I. S., Tanner, A. R., Saunders, P. K., Milluard-Sadlor, G. H., and Wright, R. 1985. Gastroenterology, 89: 1114-1122);
(11) in protecting kidneys against acute pyelonephritis (Robert, J. A., Roth, J. K. Jr., Domingue, G., Lewis, R. W., Kaack, B., Baskin, G. 1982. J. Urology, 128: 1394-1400) and nephrotoxic nephritis in rats (Rehan., A., Johnson, K. J., Wiggin, R. C., Kunkel, R. G. and Ward, P. A.1984. Lab. Invest. 51: 396-403);
(12) in ameliorating the functional and morphological abnormalities caused because of high blood pressure (Kontos, H. A. 1985. Circ. Res. 57: 508-516);
(13) in protection against diabetes mellitus and diabetogenic activity of alloxan (Grankvist, K., Marklund, S., Sehlin, J. and Taljedal, I. B. 1979. Biochem J. 782: 17-24.);
(14) in protection of tracheal cells against asbestos (Mossman, B. T., and Landesman, J. M. 1983. Chest, 835: 50s-51s);
(15) in relieving the depressor effect of spinal cord injury (Taoka, Y., Urakado, M., Koyanagi, E., Naruo, M., Inoue, M. In Frontiers of Reactive Oxygen Species in Biology and Medicine (Eds. Asada, K. and Yoshikawa, T.) Excerpta Medica, Amsterdam, pp. 241-242).
When SOD is to be injected in the body, a sterile composition would be needed and for that an autoclavable SOD would be an ideal one. Moreover, in reperfusion applications and storage of organs at low temperature, an autoclavable SOD would be required which can function efficiently at low temperature as well. Apart from the use of autoclaved SOD in pharmaceuticals and medical fields, sterile SOD will also be a choice in the cosmetic and food industry (for preventing oxygen disorders) as well.
(a) A number of formulations have been developed for pharmaceutical and cosmetic applications using SOD as one of the important antioxidant ingredients to scavange oxy free radicals from the system where applied. Availability of a SOD with autoclavability to ensure a germ free sterile preparation [the maximum thermostability of SOD described so far is at 80° C. (Gudin; Claude; Trezzy; Claudine 1996. U.S. Pat. No. 5,536,654); Bonaccorsi di Patti, M. C., Giartosio, A., Musci, G., Carlini, P. and Calabrese, L. (In Frontiers of Reactive Oxygen Species in Biology and Medicine. 1994. (Eds. Asada, K. and Yoshikawa, T.), Excerpta Medica, Amsterdam, pp. 129-130)], stability without adding an external stabilizer [the addition of hydrogen peroxide trapping agent, polyols, and sugars etc. are required to stabilise the enzyme from other sources such as germinated plant seeds (Bresson-Rival; Delphine; Boivin; Patrick; Linden; Guy; Perrier; Erric; Humbert; Gerard; 1999; U.S. Pat. No. 5,904,921)] and a wide range of temperature functionality from sub-zero to above 50° C. temperature [temperature range for SOD activity has been reported between 5 to 45° C. most of the workers (Burke, J. J. and Oliver, M. J. (Plant Physiol. 1992. 100: 1595-1598); Hakam, N. and Simon, J. P. (Physiol. Plant. 1996. 97: 209-216)] would immensely enhance the utility of the formulations and be more safe for use for humans.
The formulations/compositions mentioned below, but not limited to those mentioned below, have included SOD as one of the active ingredients:
(a)Hersh, T. in U.S. Pat. No. 5,922 dated Jul. 13, 1999 disclosed a composition for ameliorating free radical damage induced by tobacco products and environmental pollutants. The composition included, as active ingredients, reduced glutathione (0.5 mg) and a source of selenium (5 &mgr;g) selected from the group consisting of elemental selenium, selenomethionine and selenocysteine. The active ingredients were combined
Ahuja Paramvir Singh
Kumar Sanjay
Sahoo Rashmita
Council of Scientific and Industrial Research
Foley & Lardner
Weber Jon P.
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